Synthesis of Ajoene Analogues by Novel Synthetic Strategies

Article abstract of DOI:10.1002/chem.202005023

Ajoene is a compound found in garlic extracts exhibiting a large range of biological activity. Novel ajoene analogues have been prepared in the search of compounds with superior bioactivity. Modifications include the alteration of the sulfoxide, the centr

Full text of DOI:10.1002/chem.202005023

Accepted Article
Accepted Article
Title: Synthesis of Ajoene Analogues by Novel Synthetic Strategies
Authors: Marina Yamamoto Raynbird, Shaista Khokhar, Daniel Neef,
Gareth Evans, and Thomas Wirth
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10.1002/chem.202005023
Chemistry - A European Journal
COMMUNICATION
Synthesis of Ajoene Analogues by Novel Synthetic Strategies
Marina Yamamoto Raynbird,[a] Shaista S. Khokhar,[b] Daniel Neef,[b] Gareth J. S. Evans[b] and Thomas
Wirth*[a]
Abstract: Ajoene is a compound found in garlic extracts exhibiting a
thesis.[3] More recently, the precursor 4 was synthesised in a flow
process and also converted to 3 (Scheme 1d).[4]
large range of biological activity. Novel ajoene analogues have been
prepared in the search of compounds with superior bioactivity. Modi-
fications include the alteration of the sulfoxide, the central alkene and
O
O
S
F
O
S
the terminal allyl groups.
S
S
F
R
S
R’
F
S
R
S
S
R’
S
5
6
7
Garlic extracts are known for a very long time to exhibit a wide
range of biological properties. Ajoene (3) is a molecule which is
formed from alliin 1 and allicin 2 as naturally occurring compounds
in garlic, and has potent antithrombotic inhibitory properties
(Scheme 1a).[1] (E/Z)-Ajoene 3 was first observed in 1983 by
Apitz-Castro[2] and was fully characterised by Block in 1984.[1]
Block and co-workers have obtained (E/Z)-3 through a thermal
rearrangement of allicin (2) (Scheme 1b). Although Block isolated
3 in 37% yield as a mixture of its geometric isomers, many other
rearrangement and decomposition products were also obtained,
including mono and polysulfides as well as vinyl-dithiins.
R
S
R’
S
S
R
S
S
N
8
9
Figure 1. Reported analogues of Ajoene 3.
In recent years there has been increased activities in the synthe-
sis of non-natural compounds based on the core structure of 3
and attempts to generate compounds with superior biological ac-
tivity (Figure 1). In 1986, Block et al. extended their work by de-
veloping ajoene analogues of type 5 containing a central aromatic
moiety.[5] In 2008 and 2012, Hunter et al. reported the synthesis
of analogues 6 with terminal end modifications.[6] More recently,
Block et al. reported the rearrangement of fluorinated allicin to
yield fluorinated ajoene 7.[7] Additionally, Fong et al. reported
ajoene analogues 8 and 9 which are based on disulfides.[8] An
aryl-substituted analogue has also been investigated.[9] Gruhlke
and co-authors have very recently reported sulfilimine analogues
of allicin.[10] Compounds 5–9 have been biologically evaluated
and compared to the activity of 3. Different assays have been
used and some derivatives have shown promising activity.
In 2018, we reported the first total synthesis of 3 (Scheme 1c).
The chemical total synthesis overcame obstacles such as the in-
stability and volatility of 2 by replacing allicin with alternative start-
ing materials to develop a reliable and robust five step syn-
a) Garlic:
O
S
O
S
Alliinase
NH2
CO2H
O
S
S
S
S
–H2O
NH2
2 Allicin
3 Ajoene
1 Alliin
–
CO2H
b) Block (1984):
O
S
O
S
S
S
O
S
Acetone/water (3:2)
reflux
S
S
2
3 (37%, E:Z 4:1)
S
S
R
O
S
O
S
S
c) Wirth (2018):
1. KOH / TsSallyl
2. H2O2
O
S
R
R
R
S
R
S
S
S
SAc
S
Type I
Type II
central alkene modification:
SePh
3 (23%, E:Z 1:1)
S
R
S
S
d) Wirth (2020):
S
O
central alkene and sulfoxide
modification:
mCPBA
S
S
S
S
Type III
S
S
3 (49%)
4
Figure 2. Library of ajoene analogues incorporating modifications at the central
Scheme 1. Synthetic routes to ajoene (3): a) Biosynthetic route in garlic. b) Syn-
thesis described by Block et al. in 1984 by thermal rearrangement of 2 in aque-
ous acetone. c,d) Synthesis of 3 by Wirth and coauthors.
alkene and the sulfoxide (Types I-III).
We will expand upon current analogues to produce a wide library
of novel ajoene analogues which are reported here. Biological ac-
tivities of all analogues have been evaluated and compared to the
parent compound ajoene 3. The first series of analogues have
incorporated a modification in the central alkene unit and at least
one terminal group R. The central alkene has been replaced by a
cyclopropyl moiety (Type I), or by an aromatic ring (Type II) in a
total of 10 examples. In the third modification (Type III) the central
alkene is replaced by an aromatic ring and a disulfide is installed
in place of the original sulfoxide (Figure 2). At present, there are
no methods to synthesise (E)-3 and (Z)-3 selectively and replac-
ing the central double bond with another moiety could lead to a
pharmaceutically relevant drug molecule. Although the selective
[a]
[b]
M. Yamamoto Raynbird, Prof. Dr. T. Wirth
School of Chemistry
Cardiff University
Park Place, Main Building, Cardiff CF10 3AT (UK)
E-Mail: wirth@cf.ac.uk
Dr. S. S. Khokhar, Dr. D. Neef, Dr. G. J. S. Evans
Neem Biotech
Roseheyworth Business Park North
Abertillery NP13 1SX (UK)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.202005023
Chemistry - A European Journal
COMMUNICATION
synthesis alkenyl disulfides has been reported, it has not been
used in the synthesis of ajoene derivatives.[11] Block observed that
(Z)-3 has a higher activity than (E)-3 and thus, a selective synthe-
sis for either isomer would be advantageous.[5] A central cyclopro-
pyl moiety was chosen to replace the central olefin as this would
significantly affect the electronic properties of the molecule, with-
out greatly affecting the steric demand of the central part. Ajoene
3 is formed as a racemate in garlic extracts and investigations on
the effect of the sulfoxide chirality on the biologic activity have yet
to be reported.
MIRC protocols using benzyl mercaptan or thiobenzoic acid were
also investigated. While the MIRC product was obtained in 42%
yield with benzyl mercaptan, the benzylic protecting group could
not be cleaved under standard conditions. By employing thioben-
zoic acid in the MIRC reaction, the thiobenzoic acid proved to be
a harder nucleophile resulting in a direct bromide substitution (see
supporting information).
We have drawn inspiration from the work by Block who synthe-
sised central aromatic analogues of type 5.[5] We prepare aro-
matic compounds where the sulfoxide and disulfide are ex-
changed. This will alter their electronic properties and the reactiv-
ity of both moieties. Eight examples with meta- and ortho-substi-
tution have been synthesised by a novel route as shown in
Scheme 3.
An eight-step route has been developed, with a Michael Induced
Ring Closure (MIRC) for the cyclopropane synthesis being the key
step. Ester 12 was obtained in 69% yield using an adapted
method from the literature,[ 12 ] which is formed exclusively as
trans-isomer. J-coupling values were in agreement in those re-
ported by Bernard, who had confirmed the trans-configuration by
2D NMR and NOSEY experiments. After formation of the central
moiety, the sulfide is installed over three steps (ii−iv), by reduction,
Mitsunobu reaction[13] and deprotection/allylation to yield 15. The
challenging step of the synthesis was the subsequent deprotec-
tion of the para-methoxy benzyl (PMB) group. The deprotection
(v) suffered from low yields due to the volatility and instability of
the intermediate thiolate. A large excess of trifluoroacetic anhy-
dride and acetic acid allowed a cleavage of the PMB group with
direct trapping of the thiolate as thioacetate 16 in 20% yield. De-
spite the PMB deprotection resulting in low yields, there is an ad-
vantage in employing such a group. The advantage of having an
acid sensitive protecting group is its stability in the previous step
(iii) using basic conditions avoiding the formation of side products.
Due to the low yield, other acid-sensitive protecting groups should
be considered in the future. With thioacetate 16 synthesised, the
disulfide bond was installed yielding 17a and 17b using a thioto-
sylate reagent as an electrophilic sulfur source. Finally, mono-ox-
idation of 17 with mCPBA furnished the desired sulfoxides 18a
and 18b (Scheme 2).
I
I
SAc
S
R
(i)
(ii)
(iii)
Br
SAc
SAc
SAc
19
20
21
22
O
S
R
S
R
(iv)
(v)
S
R’
S
R’
S
S
23
24
Scheme 3. Synthesis of ajoene analogues 24. Conditions and reagents: (i):
AcSH (1 eq.), K2CO3 (1 eq.) THF, r.t., 4 h, >95% yield; (ii): CuI (0.2 eq.), phe-
nanthroline (0.2 eq.), KSAc (1.5 eq.) in toluene 100 °C, >95% yield; (iii): K2CO3
(1 eq.), R-Br (2 eq.) in MeOH at 0 °C, 8–86% yield; (iv): KOH (1.5 eq.), R1-STs
(2 eq.) in MeOH at 0 °C, 51–88 % yield; (v): mCPBA (1 eq.) in CH2Cl2 at −78 °C,
40–78% yield. R = a: allyl, b: benzyl, c: isobutyl, d: methyl cyclopropyl.
Compound o-/m-24 can be synthesised in a concise synthetic
route starting from 2-iodobenzyl bromide o-19 or 3-iodobenzyl
bromide m-19, respectively. Employing a nucleophilic substitution
of the labile bromide in the benzyl position the first thioacetate can
be installed providing 20 in up to 99% yield. A copper(I) catalysed
coupling between aryl iodides and potassium thioacetate[14] was
employed to furnish 21 in very high yields. Since 21 has two ace-
tate protecting groups, the first deprotection uses a mild base
which cleaves the more labile acetate in the phenylic position with
some selectivity in up to 86% yield. Cleaving the acetate will un-
mask the thiophenolate which will react with an alkyl bromide as
electrophile to obtain thioether 22. Treatment of 22 with potassium
hydroxide as base cleaves the second acetate. The thiolate gen-
erates a disulfide with the thiotosylate reagent as electrophilic sul-
fur source to yield 23 in up to 85% yield. With 23 containing a
disulfide and a sulfide moiety, the addition of recrystallised
mCPBA at low temperatures enables the selective oxidation
which proceeds quickly and cleanly to yield derivatives 24 in up
to 79% yield. Different substituents R (allyl, benzyl, iso-butyl, me-
thyl cyclopropyl) were investigated to cover a spectrum of elec-
tronic and steric factors (Scheme 3).
HS
Br
(i)
(ii)
HO
+
SPMB
MeO2C
10
MeO2C
SPMB
OMe
(iv)
11
12
13
(iii)
(v)
AcS
SPMB
SPMB
SAc
S
S
14
15
16
(vi)
(vii)
S
S
S
R
S
R
S
S
O
17a R = allyl
17b R = methyl cyclopropyl
18a R = allyl
18b R = methyl cyclopropyl
Scheme 2. Synthesis of ajoene analogues 18. Conditions and reagents: (i): n-
BuLi (1.1 eq.) in THF at −40 °C, 69% yield; (ii) lithium aluminium hydride (1.1
equiv.) in THF at −40 °C, 85% yield; (iii): PPh3 (2 eq.), diisopropylazodicarbox-
ylate (2 eq.), thioacetic acid (3 eq.) in THF at −20 °C, 92% yield; (iv): KOH (2.5
e.), allylbromide (2 eq.) in MeOH, −78 to −40 °C, 54% yield; (v): trifluoroacetic
acid anhydride (5 eq.), acetic acid (5 eq.) in CH2Cl2 at –78 °C, 20% yield; (vi):
allyl/methyl cyclopropyl thiotosylate (2 eq.), KOH (2 eq.) in MeOH, −78 to −40 °C,
23–27% yield; (vii): mCPBA (1.1 eq.) in CH2Cl2 at −78 °C, 14-21% yield.
Disulfides are well known to possess biological activity. This is
often the result of the instability of the disulfide bond and the sub-
sequent interaction of the thiolate with cystine residues in proteins.
[15]. We therefore synthesised a range of bis-disulfides with a cen-
tral aromatic moiety. Analogues of Type III (Figure 2) are 1,3-di-
substituted benzene derivatives which were developed to mimic
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10.1002/chem.202005023
Chemistry - A European Journal
COMMUNICATION
ajoene with E-geometry, whereas 1,2-disubstitued benzene de-
rivatives were prepared to mimic ajoene with Z-geometry. Their
synthesis is summarised in Scheme 4.
substituents. This observation is not replicated in type III ana-
logues, suggesting there is an interaction between the benzyl and
sulfoxide group (Table 2, entries 4–11). Interestingly, for bis-disul-
fide compounds of type III 25a–d, the MBIC is significantly lower
in the cases of ortho-compounds over the corresponding meta-
compounds against S. Aureus and where o-25a, o-25c, o-25d all
gave superior MBIC values in comparison to ajoene. This shows
the importance of the disulfide moiety and suggests that the ter-
minal group has a great influence on its mode of action, where a
more sterically hindered disulfide shows higher biological potency
(Table 2, entries 12–23). A standout result can be seen by both
m-25a and o-25a, which also shows impressive MBIC values in
comparison to ajoene (3). Although m-25a inhibits biofilm produc-
tion in S. Aureus, the result of particular interest is seen against
P. Aeruginosa. Here, the MBIC value is substantially lower, sug-
gesting that analogue m-25a is up to 30 times more potent than
3. Out of the 22 substrates tested, it was only o-24b and m-25a
that gave an MBIC value of single digit magnitude against P. Ae-
ruginosa (Table 2, entry 12).
S
S
R
(i)
(i)
S
R
S
S
S
m-21
o-21
S
R
R
S
m-25
o-25
Scheme 4. Synthesis of ajoene analogues 24. Conditions and reagents: (i):
KOH (3 equiv.), R-thiotosylate (4 eq.) in MeOH at 0 °C, 1–98% yield.
The bis-disulfides 25 could be obtained by cleaving both thioace-
tates in 24 with a strong base and reacting the thiolate with a thi-
otosylate as an electrophilic sulfur source. The efficiency and
ease of this step (i) varied greatly depending on the thiotosylate
employed in the reaction. A general trend shows that allyl and al-
kyl substituents gave higher yield than benzyl substituents. The
yield suffered significantly when a strong electron withdrawing or
donating group was attached to the para-position of the benzyl
substituent. Generally, yields were higher for the m-25 than o-25,
presumably due to the lower steric hinderance. The low yields for
m-25f-h was a result of difficulties during purification, as each
compound required normal phase chromatography followed by
reverse phase separation (Table 1).
Table 2. Biological evaluation of type I analogues (entries 2−3), type II ana-
logues (entries 4−11) and type IIII analogues (entries 12−23) in comparison to
ajoene (3, entry 1) against S. Aureus and P. Aeruginosa bacteria.
O
R
S
R
S S
S
S
R
S
S
R’
S
R
O
S
S
Table 1. Yields for the synthesis of novel unsymmetrical bis-disulfides 25.
18
24
25
S. Aureus
P. Aeruginosa
Entry
Compound
Entry
1
Compound
m-25a
m-25b
m-25c
m-25d
m-25e
m-25f
Substituent R
allyl
Yield [%]
69
MBIC IC50 (µM)
0.565
>48
MBIC IC50 (µM)
1
2
3
49.2
>128
131
2
benzyl
21
18a
3
iso-butyl
81
3
18b
>48
4
methyl cyclopropyl
4-methyl benzyl
4-CF3-C6H4-CH2
4-OCH3-C6H4-CH2
4-(CO2CH3)-C6H4-CH2
allyl
98
4
m-24a
m-24b
m-24c
m-24d
o-24a
o-24b
o-24c
o-24d
m-25a
m-25b
m-25c
m-25d
m-25e
m-25f
m-25g
m-25h
o-25a
o-25b
o-25c
o-25d
9.08
0.92
8.28
7.9
69
5
70
5
>128
36.3
113
6
30
6
7
m-25g
m-25h
o-25a
15
7
8
1
8
12.5
4
>128
8.3
9
48
9
10
11
12
o-25b
benzyl
41
10
11
12
13
14
15
16
17
18
19
20
21
22
23
14.5
16.2
0.223
36
54.8
>128
1.64
>144
>144
>144
>128
>128
>128
>128
15.8
63.4
>128
65.5
o-25c
iso-butyl
65
o-25d
methyl cyclopropyl
55
In total, 22 novel analogues were biologically evaluated by Mini-
mum Biofilm Inhibitory Concentration (MBIC) studies using S. Au-
reus and P. Aeruginosa bacteria (Table 2). For compounds 18a
and 18b, the MBIC for S. Aureus was at least 85 times higher than
for ajoene (3). This suggests that the central olefin in 3 plays an
important role in its mode of action, one which the central cyclo-
propyl cannot replicate. A similar pattern is seen for P. Aeruginosa
where analogue 18a and 18b showed MBIC values at least over
2.6 times higher. Therefore, the potency of 18a–b analogues is
significantly reduced with the central cyclopropyl modification (Ta-
ble 2, entries 1–3). Type II analogues show a more complex pat-
tern where the MBIC results suggest that there may be several
factors at play. In general, o-24a–d showed higher MBIC results
than m-24a–d. In both the ortho- and the meta-analogues, a ben-
zyl terminal group shows significantly superior potency against S.
Aureus in comparison to allyl, iso-butyl and methyl cyclopropyl
42
44
5.6
17
7.2
1.3
0.172
1.4
0.48
0.425
In conclusion, 22 substrates of novel ajoene analogues have been
synthesised over three novel routes. These analogues have been
biologically evaluated in their potency to inhibit the biofilm produc-
tion of two bacteria. Promising results were found in the MBIC
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10.1002/chem.202005023
Chemistry - A European Journal
COMMUNICATION
value of o-25a against S. Aureus and m-25a against P. Aeru-
ginosa which showed superior potency of 3 and 30 times greater
than ajoene, respectively.
Knowledge Economy Skills Scholarships (KESS) is a pan-Wales
higher level skills initiative led by Bangor University on behalf of
the HE sector in Wales. It is part funded by the Welsh Govern-
ment’s European Social Fund (ESF) convergence programme for
West Wales and the Valleys.
Acknowledgements
We thank Neem Biotech Ltd., especially Danielle Williams, and
Lucy Andrews for the biological assays and the KESS 2 program
(MYR) and the School of Chemistry, Cardiff University, for finan-
cial support. We thank James Silk and Alexander Lees, School of
Chemistry, Cardiff University, for the synthesis of intermediates.
Keywords: ajoene • biologic activity • disulfides • garlic • or-
ganosulfur compounds
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COMMUNICATION
Non-natural Allium analogues of three types have been synthesised
Marina Yamamoto Raynbird,
Shaista S. Khokhar, Daniel Neef,
Gareth J. S. Evans, Thomas Wirth*
by novel synthetic strategies. Analogues have been biologically evalu-
ated in their ability to inhibit biofilm production in S. Aureus and P. Aeru-
ginosa with promising results.
Page No. – Page No.
Synthesis of Ajoene Analogues
by Novel Synthetic Strategies
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